experimental manipulations of early drosophila...

/ . Embryol. exp. Morph. Vol. 32, 1, pp. 273-285, 1974 2 7 3

Printed in Great Britain

Experimental manipulations of earlyDrosophila embryos

II. Adult and embryonic defects resulting from theremoval of blastoderm cells by pricking

By MARY BOWNES1 AND J. H. SANG2

From the School of Biological Sciences, University of Sussex

SUMMARYCells were removed from three regions (anterior, mid-lateral and posterior) of Drosophila

eggs at blastoderm formation, by pricking. Embryonic defects were generally correlated withthe site of damage, as is also found with microcautery and with u.v. irradiation. Butmany structures can develop autonomously, independently of surrounding damage. Mosthatching adults were normal, suggesting that there may be mechanisms which permit com-pensation for cell loss. Malformed or missing adult structures are those expected from thefate map obtained from studies of genetic mosaics.

INTRODUCTION

It has been assumed for some time that the Drosophila egg is mosaic at leastat, if not prior to, blastoderm formation (Wigglesworth, 1939). If this is true,each cell should form a specific part of the embryo or adult, and if cells aredamaged or removed the relevant parts should be missing. Howland & Child(1935) reported some correlation between the site of pricking blastoderm eggswith particular defective structures found in adults. The age of their eggs wasrather variable, and as very few adult defects were found their evidence cannot beused as proof of determination at this stage. Howland & Sonnenblick (1936),on the other hand, found some evidence of regulation in nuclear multiplicationstage Drosophila embryos after removal of material by pricking. The implicationwas that determination was established in the uniform cellular blastoderm, butnot before. The subject has now been pursued using this simple technique.

The experiments described in this paper were designed to test the idea ofmosaic determination in blastoderm eggs of Drosophila with respect to bothlarval and adult organization. The important difference between this techniqueand u.v. irradiation (Hathaway & Selman, 1961; Nothiger & Strub, 1972;

1 Author's address: Center for Pathobiology, University of California, Irvine, California92664, U.S.A.

2 Author's address: School of Biological Sciences, University of Sussex, Falmer, Brighton,Sussex BN1 9QG, U.K.

l8 EM B 32

274 M. BOWNES AND J. H. SANG

Bownes & Kalthoff, 1974) or microcautery (Bownes & Sang, 1974), which havebeen used to damage regions of the egg, is that cells are physically removed fromthe blastoderm. Damaged molecules may be repaired by mechanisms within theegg after u.v. irradiation (Setlow, 1966) or possibly after microcautery; but ifcells are actually removed and normal larvae and adults are produced, then cellsin the surrounding area must be capable of compensating for the absent cells,and regulation of some type is occurring.

Pricking experiments can show that determination for adult structures hasoccurred if damage to areas known from somatic cross-over studies to containpresumptive adult cells (Hotta & Benzer, 1972) consistently produce defects inthe expected adult structures. However, if defects are not found it does notprove that the areas were not determined at this time. Schubiger (1971) hasshown that the discs of 3rd instar larvae are capable of regenerating the missingparts when they are cut into portions, so long as a specific area of the disc isleft intact. It seems likely that this could also be true at earlier stages, so thechances of damaging or removing all the cells necessary to produce an adultwith defective cuticular structures may be quite small. Another possibility is thata small group of cells may be determined to be an adult disc, but at this stageremoval of any part of them does not result in a defective adult, due to compen-sation for the loss by surrounding cells. It is probable, too, that many surround-ing larval cells will be removed, the changes of embryonic or larval death will beincreased, and adults may rarely emerge. But larval defects can then be identified.

Three areas of the embryo were pricked - the anterior, posterior and mid-lateral areas shown in Fig. 1. From the data, resulting embryonic defects couldbe compared wih those found after partial u.v. irradiation of Drosophila embryosat blastoderm formation (Bownes & Kalthoff, 1974), and the expected adultdefects could be predicted from Fig. 2.

If the egg is fully determined at blastoderm formation, some larval cellsshould always be removed and all treated eggs should produce defective embryosor larvae, even if these cannot always be identified. As the number of disc cellsis in all cases less than the number of cells removed (Nothiger, 1972), adultdefects should be found, provided the larvae survive. The probability of pro-ducing adults with abnormal heads should be high after pricking the anteriorregion; mid-region damage should cause thoracic deficiencies, and posteriortreatments would be expected to cause abdominal defects.

MATERIALS AND METHODS

Origin and preparation of eggs

All eggs were of Oregon K stock and were collected on agar plates coated witha paste of yeast and sugar. Egg collections were made every 30 min (after thefirst hour of laying, in order to exclude any partially developed retained eggs).Eggs were then left to develop for a further 45 min, before being washed from the

Drosophila defects after blastoderm cell removalANTERIOR

275

VENTRAL

Anterior Head

Middle

Legl

Leg 2

Leg 3

Posterior

Proboscis

Thorax

Wing DORSAL

— Abdomen

POSTERIOR

Fig. 1 Fig. 2

Fig. 1. Location of regions where cells were removed for these experiments.

Fig. 2. Location of presumptive adult disc cells. This figure is based on the map pro-duced by Hotta & Benzer (1972) using genetic mosaic studies. The egg is 0-42 mmlong.

agar with 0-9 % sodium chloride, then dechorionated with 3 % sodium hypo-chlorite for 5 min. They were washed in isotonic sodium chloride, and eggs atthe cellular blastoderm stage of development were selected, using a dissectingmicroscope.

Experimental procedureEggs of the correct developmental stage were placed on a piece of black filter

paper, as this makes the eggs easily visible for orientation and also dries them.If the surface of the egg is damp, pricking causes the egg to collapse completelyas the contents drain out by capillary action. However, with slight drying theglobule of cells remains on the surface of the egg (Fig. 3). The eggs were prickedwith a fine tungsten needle mounted in a glass holder. The needle was made therequired shape by electrolytically removing some of the tungsten at the tip insodium hydroxide; suitable needles were chosen by trial. The pricked eggs werethen transferred on the filter paper to small dishes, and covered in liquid paraffin.The dishes were placed in damp sealed boxes at 25 °C for 24 h.

Scoring

The eggs were classified into (1) normal larvae, (2) undifferentiated eggs whichfailed to continue development after pricking and (3) defective embryos.

18-2


Fig. 3. Typical appearance of an egg after pricking at blastoderm stage, with a smallgroup of cells on the surface.

Analysis of defective embryos

Defective embryos were mounted in paraffin oil on a cavity slide, and coveredby a coverslip to prevent the eggs from collapsing further. These eggs werelooked at under higher magnification using Nomarski optics and classifiedaccording to the damaged structures. Photographs taken under these conditionsare very unsatisfactory due to the collapsed vitelline membrane and the paraffinoil, but detail within the eggs could be seen by refocusing the microscope.

Classification of embryonic defects

Class I. These embryos show only anterior defects. Mouthparts or other headstructures may be abnormal or absent, and the gut may be extruded at the anteriorof the embryo. Anterior abdominal segments may also be lacking, but theposterior end of the abdomen including the spiracles was formed by allembryos in this class.

Class II. Embryos without definite anterior or posterior specificity have beenpooled in this class. They show yolk patches and contracting masses of gut tissue;some have formed irregular bristle rows on the surface.

Class III. This class comprises all embryos with clearly posterior defects. Thegut may be extruded at the posterior end of the embryo, abdominal segments maybe partially formed or replaced by a mass of yolk and gut. All embryos in thisclass show head structures, and at least some indication of mouthparts.

Drosophila defects after blastoderm cell removal 277

Table 1. General results after removal of blastoderm cellsfrom Drosophila eggs

Eggs failing to continue developmentLarvae hatchedAbnormal embryosLarvae dyingPupae failing to hatchNormal adults hatchedAbnormal adults hatched

Total number of eggs treated

Region

Anterior

50-618031-4150—2-9—

472

damaged by pricking

Middle

64-412-52301000-32 00-3

312

Posterior

57-123-419-7151—7-10-7

264

Analysis of adults

The hatched larvae were put into vials containing yeasted Lewis medium andkept at 25 °C to continue development. Adults were scored for abnormalitiesin external morphology, and any pupae failing to hatch were dissected andabnormalities noted. Adults were kept at least 5 days to ensure that they did notdie early due to deficiencies in the soft internal parts of the gut; their fertilitywas checked, and they were then dissected to note any abnormalities of the ovaryor testis.

RESULTS

Table 1 shows that many eggs treated in each of the three regions failed tocontinue development. These eggs usually collapsed because too many cellswere removed from the embryo. Some defective embryos which failed to hatchand some larvae of apparently normal morphology also resulted from treatmentof each area. Many of these larvae died, but in all cases some adult flies werefound.

Embryonic data

The occurrence of apparently normal adults and larvae from eggs damaged atthree different areas shows that embryonic cells are capable of limited regulation,and that surrounding cells may be able to compensate for the loss of nearbyembryonic cells.

Table 2 shows the distribution of embryonic abnormalities resulting fromdamaging different regions of the egg. The classification scheme is the same asthat used for the results of irradiating different regions of the blastoderm eggwith u.v. by Bownes & Kalthoff (1974). It can be seen from Table 2 that thedistribution of embryonic defects obtained was very similar to that found afteru.v. irradiation of these areas. Anterior pricking produced anterior defects, and


Table 2. Classification of defective embryos after removalof cells by pricking and u.v. irradiation

A. Pricked eggs

Area damagedby pricking

AnteriorMiddlePosterior

Totalnumberdefectiveembryos

1487251

B. Irradiated eggs (data

Area damagedby u.v.

irradiation

Anterior quarterMiddle quarterPosterior quarter

Totalnumber

defectiveembryos

614340

I

53-543040

Distribution intoclasses (%)

A

II

46-555-5550

III

1-5410

of Bownes & Kalthoff, 1974)

I

9517004-7

Distribution intoclasses (%)

II

4-930020-9

III

74-4

posterior damage produced posterior defects. At all sites a significantly greaternumber of the more damaged, less specific Class II embryos were found afterpricking experiments than after u.v. irradiation. As with irradiation of a centralband of the egg, pricking in the mid-lateral region led to essentially anteriorabnormalities (there was no significant difference between the distribution ofabnormalities after pricking at the anterior or the mid-region, at the 5 % confi-dence level using a x2 test). The variation of defects within the classes is the sameas that described and illustrated with photographs by Bownes & Kalthoff(1974). These results corroborate the u.v. data, and suggest that there is a generalanterior/posterior organization present in the egg at blastoderm formation, butthat regulation within these areas is still possible.

Apart from the general variation of embryonic defects within the classificationused, observation of specific eggs can help our understanding of the organizationof the egg at this stage in development.

Figure 4 shows how eggs with extremely collapsed membranes, which haveobviously lost a large number of cells, are able to develop to advanced stages ofembryogenesis. This egg, which was pricked in a mid-lateral region, has developedall the abdominal segments. It has fully formed spiracles, and Malpighiantubules are present at the posterior. The head has failed to form at the anterior.Figure 5 shows an embryo, pricked at the posterior, with a normal head and all theabdominal segments formed in the anterior half of the egg, with gut extruded atthe posterior. Tracheal tissue has formed in this area. The embryo in Fig. 6 waspricked at the anterior and shows that, conversely, an embryo formed in the


Fig. 4 Fig. 5

Fig. 4. The vitelline membrane of this embryo has collapsed considerably due to lossof cells and yolk, yet some larval differentiation has occurred, including abdominalsegmentation, and formation of spiracles and Malpighian tubules. A, Anterior.Fig. 5. An embryo resulting from posterior pricking. The head and mouthparts (in)are normal and parts of all eight abdominal segments are present in the anterior halfof the egg. The posterior contains a yolk mass (y), with trachea (t) above. A, Anterior.

posterior two-thirds of the egg has all the abdominal segments and someabnormal thoracic and head formation; the rest consists of a mass of undiffer-entiated tissue.

The two types of embryo illustrated in Figs. 5 and 6 suggest that reorganiza-tion of the embryo is possible after blastoderm cells have been removed bypricking, as most of the normal structures of the embryo are formed, occupyinga smaller area than usual; much undifferentiated tissue is present, which mustnormally have a function in embryonic development.

One unique embryo (Fig. 7) resulted from pricking the mid-region of the egg.Instead of destroying basically anterior structures, as is usually seen, this embryois damaged in the middle. The embryo shows segmentation that is well developedalong the dorsal edge. It has a complete cephalo-pharyngeal complex situated


Fig. 6. An embryo resulting from anterior pricking. The posterior is well formed,having all the abdominal segments; there has been some abnormal head formation,but a large yolk patch occupies the anterior third of the embryo. P, Posterior.

obliquely at the anterior of the embryo, and has spiracles with tracheal tracts atthe posterior. Ventrally, and to the extreme anterior and posterior of the embryo,the egg contains a mass of undifferentiated tissue. It seems in this case that theegg has reacted in a more determined fashion, suggesting that the mid-regionof the anterior-posterior axis had become fixed. This occurred only once, butmay suggest that fixation of the mid-region occurs at a time close to formationof the blastoderm.

Figures 8 and 9 show that once certain structures have become determined theyseem to be able to differentiate amongst a mass of undifferentiated tissue; theydo not need to be in their correct position, nor do they need the presence oforgans normally closely associated with them in development. Figure 8 showsdetail from an egg containing nothing but some dorsally situated spiracles, andFig. 9 shows a few segments radiating from a central point over a mass ofundifferentiated tissue. Thus at least some determined cells may differentiatewhatever their locus.

Adult defects

Three of the four adult defects found were correlated with the sites of damageindicated in Fig. 1. No abnormalities were found in flies damaged in the anteriorregion. In one pupa which failed to hatch, the wing, thorax and one leg were alldefective; this embryo had been pricked in a mid-lateral region where themap indicated that these cells are located. Another embryo damaged in thisregion had a smaller defect in the meta-thoracic leg. One of the eggs pricked inthe posterior region produced an adult with an abnormally shaped abdomen.Another adult which hatched had no abnormalities in external morphology, but

Drosophila defects after blastoderm cell removal

A

281

Fig. 7. An exceptional embryo resulting from mid-region pricking. The anterior haswell formed mouthparts (m) and the posterior contains spiracles (s). Segments areformed along one side of the embryo, yet are broken on the other side where yolkhas escaped. Yolk (y) occupies one side of the embryo and part of the anterior andposterior regions of the egg. A, Anterior.

died only one day after hatching, and probably had an abnormality in the internalstructures. Abnormalities occurring with this low frequency cannot prove thatthese areas of the egg are determined at this stage in development, but theoccurrence of several normal adults from each area, when the probability ofdamaging or removing the presumptive disc cells is very high, suggests that ifthe cells are determined, there is some regulative capacity within the determinedarea.


Fig. 8. An embryo in which spiracles have formed in the absence of otherdifferentiated tissues. D, Dorsal.

Fig. 9. An embryo in which rows of segmental bristles (b) radiate out from one point,in the absence of other differentiated tissue. P, Posterior.


DISCUSSION

Perhaps the most surprising result of these experiments is that most of theadults formed after pricking in all of the three regions are apparently normal,contrary to expectation. Howland & Child (1935), Howland & Sonnenblick(1936), llmensee (1972) and Nothiger & Strub (1972) all similarly reported ahigh proportion of hatched normal adults after damaging various regions of theegg. There could be several reasons why defects are not formed. (1) Not allstructures need be present and complete to form a viable larva or adult (e.g.paired organs or large organs like fat body, etc.). Defects of this class could arisefrom hemi-lateral pricking, and would not necessarily be detected. However,this should be an exceptional class with respect to adult external morphology,and is unlikely to be a general explanation of the high adult survival, if onlybecause all regions of the egg have been damaged in the experiments noted aboveand all still give normal adults. (2) Cells may not be determined at the time oftreatment, and a more or less normal pattern of determination may be estab-lished subsequently. Chan & Gehring's (1971) experiment would argue againstthis since it shows that adult disc cells are determined in the blastoderm, andthat this determination can survive disaggregation of anterior (or posterior)halves of blastoderm eggs, their reaggregation, and transplantation and growthin adults. Bownes & Sang (1974) have shown that this determination is specificfor each disc. (3) There may be cellular regulation, as is found in the regenerationof missing parts of larval discs (Schubiger, 1971), and this seems the most likelyexplanation of these results.

Another interesting observation is the rare occurrence of head defects afterexperimental damage. Only one was found by Bownes & Sang (1974) aftermicrocautery. Nothiger & Strub (1972) found 9 head defects, but 37 thoracicand 116 abdominal defects. And in a most comprehensive survey, using u.v.irradiation of entire dorsal or ventral surfaces of eggs at various stages of develop-ment, Levin (1971) found 18 head defects, but 110 defects of thorax and scutel-lum, 245 wing defects, 175 leg defects and 625 abdominal defects. Unfortunately,Levin's dosages of u.v. vary with embryonic age (to give a constant mortality),so it is difficult to do more than note this phenomenon and its indication ofdifferential sensitivity of different discs.

The types of embryonic defects found after pricking correlate well withexperimental damage by u.v. irradiation (Bownes & Kalthoff, 1974) and bymicrocautery (Bownes & Sang, 1974), indicating the extent of determination incomparable areas. As we have noted, small areas frequently show a high degreeof autonomy, and develop independently of the damaged systems around them.This might be expected from the cell culture studies of Shields & Sang (1970;and unpublished observations) where the cells from disaggregated 4-6 h embryosform the majority of cell and tissue types in vitro. That is, the embryonic cellsshow a high degree of determinacy at this stage and proceed to their final


differentiated state; they would similarly be expected to do so in thedamaged embryo.

We would attach some weight to the positive results, where damage to adultstructures is precisely that which would be expected from genetic mappingexperiments (Hotta & Benzer, 1972). Like the mapping experiments, these resultsdo not tell us exactly when cells become determined to be adult structures. Ourearlier results (Bownes & Sang, 1974) suggest that this is not at pre-blastodermstages, which implies that the egg is not mosaic, in the sense that deter-minants of differentiation are already patterned in some final form in the eggcortex. Subsequently, determination is a progressive and developmental processwhich rapidly seems to reach a state which defines at least larval structures, andthe allocation of characteristics of particular discs to other cells. These disc cellsmultiply in the larva, and their progeny also go through a series of determinativesteps which settle their fate within a particular structure (e.g. leg) so as to give itits final form (see Nothiger, 1972). Thus, determination is a sequential, epigeneticphenomenon which we define experimentally and somewhat arbitrarily. It doesnot follow that cortical structure and content are not of primary importance forthis process: our experiments tell us nothing about this. What they do imply isthat determination of adult disc cells is established during a very short space oftime after blastoderm formation, and that there may be some regulation if theegg is damaged then. This would argue that the distinction between mosaic andregulative eggs is one of degree rather than of kind.

This work was supported by a Medical Research Council Studentship to Mary Bownes, andthe support of the Science Research Council is gratefully acknowledged. We would like tothank Mrs J. Atherton for drawing Figs. 1 and 2.

REFERENCES

BOWNES, M. & KALTHOFF, K. (1974). Embryonic defects in Drosophila eggs after partial u.v.irradiation at different wavelengths. / . Embryol. exp. Morph. 31, 329-345.

BOWNES, M. & SANG, J. H. (1974). Experimental manipulations of early Drosophila embryos.I. Adult and embryonic defects resulting from microcautery at nuclear multiplicationand blastoderm stages. / . Embryol. exp. Morph. 32, 253-272.

CHAN, L. N. & GEHRING, W. (1971). Determination of blastoderm cells in Drosophila melano-gaster. Proc. natn. Acad. Set'. U.S.A. 68, 2217-2221.

HATHAWAY, D. S. & SELMAN, G. G. (1961). Certain aspects of cell lineage and morphogenesisstudied in embryos of Drosophila melanogaster with an ultra violet micro-beam. / . Embryol.exp. Morph. 9, 310-325.

HOTTA, Y. & BENZER, S. (1972). Mapping of behaviour in Drosophila mosaics. Nature, Lond.240, 527-535.

HOWLAND, R. B. & CHILD, G. P. (1935). Experimental studies on development in Drosophilamelanogaster. I. Removal of protoplasmic materials during late cleavage and early embryonicstages. / . exp. Zool. 70, 415^27.

HOWLAND, R. B. & SONNENBLICK, B. P. (1936). Experimental studies on development inDrosophila melanogaster. II. Regulation in the early egg. / . exp. Zool. 73, 109-125.

ILMENSEE, K. (1972). Developmental potencies of nuclei from cleavage, preblastoderm andsyncitial blastoderm transplanted into unfertilized eggs of Drosophila melanogaster. WilhelmRoux Arch. EntwMech. Org. 170, 267-298.

Drosophila defects after blastoderm cell removal 285LEVIN, V. L. (1971). On the morphoses arising in Drosophila melanogaster after ultraviolet

irradiation of embryos of different ages. Tsitologiya 13, 895-905.NOTHIGER, R. (1972). The larval development of imaginal discs. In Results and Problems in

Cell Differentiation, vol. 5 (ed. H. Ursprung & R. Nothiger), pp. 1-34. Berlin: Springer-Verlag.

NOTHIGER, R. & STRUB, S. (1972). Imaginal defects after u.v. microbeam irradiation of earlycleavage stages of Drosophila melanogaster. Revue suisse Zool. 79, 267-279.

SCHUBIGER, G. (1971). Regeneration, duplication and transdetermination in fragments of theleg disc of Drosophila melanogaster. Devi Biol. 26, 277-295.

SETLOW, J. K. (1966). The molecular basis of biological effects of ultraviolet radiation andphotoreactivation. Curr. Top. Radiat. Res. 2, 195-248.

SHIELDS, G. & SANG, J. H. (1970). Characteristics of five cell types appearing during in vitroculture of embryonic material from Drosophila melanogaster. J. Embryol. exp. Morph. 23,53-69.

WIGGLESWORTH, V. B. (1939). The Principles of Insect Physiology. London: Methuen.

{Received 14 January 1974)

experimental manipulations of early drosophila...

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